Study On Mechanism Of Apoptosis Of Human Hepatocellular Carcinoma Cell Line Bel-7402 Induced By Antisence Human Telomerase RNA(hTR) Gene

Objective:To investigate the expressions of apoptosis related genes in human hepatocellular carcinoma cell line Bel-7402 transfected by antisense human telomerase RNA(hTR). To explore the apoptotic mechanism of hepatocellular carcinoma cell line Bel-7402 induced by hTR.Methods:The research team preliminary got the recombinant retroviral vector plasmid PLXSN-hTR, and then sense and antisense hTR gene were transfected into the packaging cells by electroporation, and the stablely transfected cells were selected with G418. The recombinant retroviral supernatant was collected and transfected into Bel-7402 cells. Cells were cultured in vitro. PCR was used to verify the integration of the hTR gene. Hoechst33258 fluorescent labeling was used to display the morphological evaluation of cell apoptosis. The expressions of p53, Bax, Bcl-2 mRNA in transfected and untransfected cells were determined by real-time fluorescent quantitative PCR, the expressions of p53, Bax, Bcl-2 protein were detected by Western blot.Results:1 The expression of hTR gene could be amplified in Bel-7402-hTR-EcoRI and Bel-7402-hTR-BamHI,but not in untransfected Bel-7402 cell.2 The apoptosis cells were strongly stained by Hoechst33258 compared with the control group.3 The expressions of p53 and Bax mRNA in the antisense hTR—treated group was significantly higher than those of the other two groups, while the expression of Bcl-2 mRNA was statistically lower,and the other two groups showed similar expression levels of p53,Bax and Bcl-2 mRNA.4 Western-blot showed that antisense hTR gene can increase the expressions of p53 and Bax protein in human hepatocellular carcinoma cell line Bel-7402(P<0.05) and decrease the expression of apoptosis related protein Bcl-2(P<0.05).Conclusion:1 The qualitative analysis of the apoptosis of human hepatocellular carcinoma cell line Bel-7402 can be made with the Hoechst33258 staining technique.2 Our investigation showed that the down-regulation of Bcl-2 gene expression and the up-regulation of Bax and p53 gene expression might be related to the apoptotic mechanism of heptocellular carcinoma cell line Bel-7402 induced by hTR.3The telomerase activity is positively correlated with the expressions of apoptosis regulatory gene Bcl-2,and negatively correlated with the expressions of p53 and Bax.